Saccharomyces cerevisiae is the primary biocatalyst used in the commercial production of “first generation” fuel ethanol from sugar based substrates such as corn, sugarcane, and sugarbeet. Second generation ethanol production, also known as cellulosic ethanol production, extends the carbohydrate source to more complex polysaccharides, such as cellulose and hemicellulose, which make up a significant portion of most plant cell walls and therefore most plant material.
Feedstocks commercially considered for second generation ethanol production include wood, agriculture residues such as corn stover and wheat straw, sugarcane bagasse and purpose grown materials such as switchgrass. The cellulose and hemicellose must be hydrolyzed to monomeric sugars before fermentation using either mechanical/chemical means and/or enzymatic hydrolysis. The liberated monomeric sugars include glucose, xylose, galactose, mannose, and arabinose with glucose and xylose constituting more than 75% of the monomeric sugars in most feedstocks. For cellulosic ethanol production to be economically viable and compete with first generation ethanol, the biocatalyst must be able to convert the majority, if not all, of the available sugars into ethanol.
S. cerevisiae is the preferred organism for first generation ethanol production due to its robustness, high yield, and many years of safe use. However, naturally occurring S. cerevisiae is unable to ferment xylose into ethanol. For S. cerevisiae to be a viable biocatalyst for second generation ethanol production, it must be able to ferment xylose.
There are two metabolic pathways of xylose fermentation that have been demonstrated in S. cerevisiae. The pathways differ primarily in the conversion of xylose to xylulose. In the first pathway, the XR-XDH pathway, a xylose reductase (XR) converts xylose to xylitol, which is subsequently converted to xylulose by a xylitol dehydrogenase (XDH). The XR and XDH enzyme pairs tested to date differ in required cofactor, NADH and NADPH, leading to difficulties achieving redox balance. The second commonly tried pathway converts xylose directly to xylulose using a xylose isomerase (XI) with no redox cofactor requirements. XIs from both bacterial and fungal systems have been successfully utilized in S. cerevisiae. Both pathways utilize the same downstream metabolic engineering: up regulation of the native xylulose kinase (XKS1) and four genes of the pentose phosphate pathway, specifically ribulose-phosphate 3-epimerase (RPE1), ribose-5-phosphate ketol-isomerase (RKI1), transaldolase (TAL1), and transketolase (TKL1) (FIG. 1). Use of the XI pathway also commonly entails deletion of the native aldose reductase gene (GRE3) to eliminate product lost to xylitol formation.
Xylose isomerases are known to have several metal ion binding sites, which allows XIs to bind metal ions such as manganese, cobalt, and magnesium. See, e.g., Chang et al., “Crystal Structures of Thermostable Xylose Isomerases from Thermus caldophilus and Thermus thermophilus: Possible Structural Determinants of Thermostability,” J. Mol. Biol 288:623-34 (1999). There is some indication that XIs may also bind iron cations (Fe+), but Fe+ is usually not the preferred or optimal divalent cation. However, intracellular iron regulation and metabolism is known to be a critical function for eukaryotic cells due to iron's role as a redox-active protein cofactor. See, e.g., Outten and Albetel, “Iron sensing and regulation in Saccharomyces cerevisiae: Ironing out the mechanistic details,” Curr. Op. Microbiol. 16:662-68 (2013). Intracellular iron levels are primarily controlled by the iron-sensing transcriptional activators Aft1 and Aft2 in S. cerevisiae. Iron-sulfur (Fe/S) clusters are essential for transcriptional control by Aft1/2 and Yap5 during iron sufficiency. Under sufficient iron levels, Fe/S clusters are synthesized in the mitochondria through the integration of iron, sulfur, and redox control pathways. The Fe/S clusters interact with Grx3, Grx4, Fra1, and Fra2 to inactivate Aft1/2, leading to down regulation of Aft1/2 target genes. Fe/S clusters also are known to activate the expression of Yap5 target genes, including CCC1. Ccc1 stimulates the import of iron and its sequestration in the vacuole.